title: Conformational dynamics of a regulator of G-protein signaling protein reveals a mechanism of allosteric inhibition by a small molecule.
abstract: Regulators of G protein signaling (RGS) proteins are key players in regulating signaling via G protein-coupled receptors. RGS proteins directly bind to the Gα-subunits of activated heterotrimeric G-proteins, and accelerate the rate of GTP hydrolysis, thereby rapidly deactivating G-proteins. Using atomistic simulations and NMR spectroscopy, we have studied in molecular detail the mechanism of action of CCG-50014, a potent small molecule inhibitor of RGS4 that covalently binds to cysteine residues on RGS4. We apply temperature-accelerated molecular dynamics (TAMD) to carry out enhanced conformational sampling of apo RGS4 structures, and consistently find that the α5-α6 helix pair of RGS4 can spontaneously span open-like conformations, allowing binding of CCG-50014 to the buried side-chain of Cys95. Both NMR experiments and MD simulations reveal chemical shift perturbations in residues in the vicinity of inhibitor binding site as well as in the RGS4-Gα binding interface. Consistent with a loss of G-protein binding, GAP activity, and allosteric mechanism of action of CCG-50014, our simulations of the RGS4-Gα complex in the presence of inhibitor suggest a relatively unstable protein-protein interaction. These results have potential implications for understanding how the conformational dynamics among RGS proteins may play a key role in the sensitivity of inhibitors.
authors.affiliation: Department of Chemical Engineering, University of New Hampshire , Durham, New Hampshire 03824, United States of America.
authors.forename: Harish
authors.initials: H
authors.lastname: Vashisth
authors.forename: Andrew J
authors.initials: AJ
authors.lastname: Storaska
authors.forename: Richard R
authors.initials: RR
authors.lastname: Neubig
authors.forename: Charles L
authors.initials: CL
authors.lastname: Brooks
mesh_terms: D000494:Allosteric Regulation; D000495:Allosteric Site; D003545:Cysteine / Q000737:chemistry* / Q000378:metabolism; D006801:Humans; D007700:Kinetics; D062105:Molecular Docking Simulation; D056004:Molecular Dynamics Simulation; D011485:Protein Binding; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D020710:RGS Proteins / Q000037:antagonists & inhibitors / Q000737:chemistry* / Q000378:metabolism; D054852:Small Molecule Libraries / Q000737:chemistry* / Q000378:metabolism; D045162:Thiazolidinediones / Q000737:chemistry* / Q000378:metabolism
chemical_list: C558674:4-(4-fluorobenzyl)-2-p-tolyl-1,2,4-thiadiazolidine-3,5-dione; D020710:RGS Proteins; D054852:Small Molecule Libraries; D045162:Thiazolidinediones; C098799:RGS4 protein; D003545:Cysteine